Glucosylceramide synthase inhibition protects against cardiac hypertrophy in chronic kidney disease

A significant population of patients with chronic kidney disease (CKD) develops cardiac hypertrophy, which can lead to heart failure and sudden cardiac death. Soluble klotho (sKL), the shed ectodomain of the transmembrane protein klotho, protects the heart against hypertrophic growth. We have shown that sKL protects the heart by regulating the formation and function of lipid rafts by targeting the sialic acid moiety of gangliosides, GM1/GM3. Reduction in circulating sKL contributes to an increased risk of cardiac hypertrophy in mice. sKL replacement therapy has been considered but its use is limited by the inability to mass produce the protein. Therefore, alternative methods to protect the heart are proposed. Glucosylation of ceramide catalyzed by glucosylceramide synthase is the entry step for the formation of gangliosides. Here we show that oral administration of a glucosylceramide synthase inhibitor (GCSi) reduces plasma and heart tissue glycosphingolipids, including gangliosides. Administration of GCSi is protective in two mouse models of cardiac stress-induction, one with isoproterenol overstimulation and the other with 5/6 nephrectomy-induced CKD. Treatment with GCSi does not alter the severity of renal dysfunction and hypertension in CKD. These results provide proof of principle for targeting glucosylceramide synthase to decrease gangliosides as a treatment for cardiac hypertrophy. They also support the hypothesis that sKL protects the heart by targeting gangliosides.


Results
Glucosylceramide synthase inhibitor reduced TRPC6 current in vitro. Pathologic cardiac remodeling occurs during times of cardiac stress. TRPC6 is an important player of this process by providing a feedforward amplification cascade. TRPC6 channels are present in lipid rafts (caveolae) of cardiac myocytes; 19 sKL exerts cardioprotection through targeting sialogangliosides and down regulates raft-associated TRPC6 activity 14,18 . We have previously shown that a GCSi, NB-DGJ (N-(n-Butyl)deoxygalactonojirimycin), inhibits TRPC6 channel similar to sKL by targeting sialogangliosides. GZ667161 is a newly developed GCSi with higher potency (Fig. 1A). Here, we first validated the effect of GZ667161 on TRPC6. As previously described, HEK293 cells expressing recombinant TRPC6 were treated with sKL and/or GZ667161. We observed that TRPC6 current density is significantly reduced when treated with GZ667161 or sKL as compared to no treatment 18 . When cells were co-treated with sKL and GZ667161 there was no additive effect on the TRPC6 current, indicating that they act in the same pathway (Fig. 1B,C). The effect of GZ667161 was due to blocking the synthesis of GM1, as addition of GM1 in GZ667161-treated cells leads to a partial restoration of TRPC6 current. Supporting the notion that sKL inhibits TRPC6 currents by targeting to GM1, we found that TRPC6 currents rescued by GM1 can be inhibited by sKL. These outcomes recapitulate results of our previous studies using NB-DGJ 14 . The potency of GZ667161 is 50-fold higher than for NB-DGJ: the maximal inhibition of TRPC6 occurs at a concentration of 0.5 μM for GZ667161 vs 25 μM for NB-DGJ 14 . Thus, GZ667161 is a good potential candidate for studying cardioprotection in in vivo models of heart failure and CKD. GSC inhibitor reduces glycosphingolipids (GSL) in the plasma and heart tissue. GZ667161 is a potent orally available small molecule inhibitor of GCS 31 . It has recently been used in pre-clinical studies as a substrate reduction therapy effecting the brain. To investigate the effect of GZ667161 on cardioprotection, we studied its effect on Isoproterenol (ISO)-induced cardiomyopathy. ISO overstimulation is a well-accepted stressinduced cardiomyopathy model 32,33 . Age matched mice were fed control diet at day -7, and then separated in two groups at day -3, one group continued on control, and the other one switched to a 0.033% wt/wt GZ667161 diet ( Fig. 2A). Thereafter, mice received subcutaneous ISO daily for 16 days. ECHO was performed on mice after the last day of ISO treatment. After 16 days of consecutive treatment of ISO, mice were euthanized, and blood and tissues were collected for analysis.
Plasma levels of total GSL were measured and normalized to total phosphatidylcholine (PC). Ceramide levels between the groups remained unchanged (Fig. 2B) and total glucosylceramide were reduced in mice treated with GZ667161 with or without ISO induction as compared to WT mice on control diet (Fig. 2C). Along with the reduction in glucosylceramide, there was a reduction in total lactosylceramide (GL2) and GM3 levels. (Fig. 2D,E). In heart tissue of WT mice treated + /-ISO and given a GZ667161 diet, ceramide levels remained unchanged, while a reduction of total glucosylceramide levels was present and met with a reduction of both total GL2 and GM3 ( Fig. 2F-I). These results confirm that treatment with GZ667161 leads to reduction of glycosphingolipids (GSL) in vivo by blocking the synthesis of glucosylceramide from ceramide. nificantly reduces GSL levels in the heart and in plasma of treated mice, we next examined its effect on heart weight/body weight (HW/BW) ratios, an index to measure cardiac hypertrophy. Mice on 0.033% GZ667161 diet showed a significant decrease in body weight (BW) compared to mice on control diet (Fig. 3A). As expected, ISO treatment significantly increased heart weight and HW/BW ratios in mice on control diet indicating hypertrophy ( Fig. 3B,C). ISO treatment also induced cardiac hypertrophy in mice fed on GZ667161 diet (" + ISO" vs "-ISO"), though the degree of ISO-induced hypertrophy was moderately reduced in GZ667161-treated vs control diet ("GZ667161 + ISO" vs "control + ISO"). Body weight loss in GZ667161-treated mice may have confounded the analysis of HW/BW ratios. GZ667161 treatment did not affect tibia length (TL) (Fig. 3D). Thus, we normalized HW to tibia length (TL) (Fig. 3E) 34 . In mice fed on control diet, ISO overstimulation increased HW/ TL, confirming cardiac hypertrophy. There were no significant differences in HW/TL between ISO-vs vehicletreated mice fed with GZ667161, indicating that GZ667161 diet ameliorated ISO-induced cardiac hypertrophy. Furthermore, left ventricular myocyte area was increased by ISO and ISO-induced increases were reduced by GZ667161 treatment (Fig. 3F). ECHO revealed that ISO treatment caused functional decline (decreases in ejection fraction) in control mice but not in GZ667161-treated mice (Fig. 3G). Therefore, along with the effect on cardiac mass, GZ667161 also prevented ISO-induced cardiac function decline. The effect of GZ667161 is not mediated through changes in blood pressured (Fig. 3H). During times of cardiac stress, cardiac remodeling and cardiac growth occurs through re-expression of fetal heart genes, such as atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), which are generally turned off at birth. The expression of TRPC6 in the normal heart is very low. However, it is upregulated during cardiac stress by the pathologically activated Ca 2+ -calcineurin-NFAT signaling cascade. Fibrosis and increased expression of collagen-I (Col-I) and collagen-III (Col-III) are consequences and hallmarks of pathological cardiac hypertrophy. We examined the effects of GZ667161 on genes associated with cardiac remodeling including cardiac fetal genes (ANP and BNP), TRPC6, and Col-I, -III by using quantitative RT-qPCR. Results revealed that ISO overstimulation in mice fed with control diet induced a significant increase in expression of ANP, BNP, TRPC6, and Col-I, -III ( Fig. 4A-E). Feeding mice GZ667161 diet, blunted the ISO-induced increases in gene   www.nature.com/scientificreports/ expression. To test the extent of fibrosis in the hearts of mice we performed a trichrome staining to measure for collagen, shown in blue. We found that GZ667161 reduced fibrosis in the hearts of mice as compared to + ISO control mice (Fig. 4F).
GZ667161 exerts cardioprotection on CKD mice and reduces CKD induced heart failure. sKL protects CKD-associated cardiomyopathy by targeting GSL gangliosides, therefore providing a basis to examine the effect of GZ667161 on CKD-induced stress and dysfunction using a 5/6 nephrectomy mouse model.
In pilot experiments we found CKD mice do not tolerate the same dose of GZ667161 as used for ISO studies (0.033% wt/wt) (not shown). Uremic environment may contribute to the reduced tolerance to GZ66716. Hence, we performed experiments testing the effect of reduced dose in our ISO overstimulation model. We first used gavage feeding to eliminate potential dosage variation from variable food intake. Gavage feeding at 20 mg/kg of GZ667161 (equivalent to 0.011% wt/wt) did not cause weight loss yet conferred cardioprotection ( Fig. 5A-C).
We found that gavage feeding 20 mg/kg significantly decreased HW/TL and expression of BNP compared control diet in ISO overstimulation model (Fig. 5B,C). We next examined the effects of dietary feeding of the reduced dose GZ667161 (0.011% wt/wt) and confirmed that this reduced dose prevented body weight loss as compared to the higher dose (0.033% wt/wt) (Fig. 5D) while conferred moderate protection against ISO-induced increases in heart mass index, BNP gene expression, Col-I expression, and fibrosis ( Fig. 5E-H). Having confirmed that 0.011% GZ667161 diet is effective in ISO-induced model, we used the reduced dose in CKD studies. www.nature.com/scientificreports/ Age-matched mice received sham or 5/6 nephrectomy procedure. Mice were switched from regular rodent chow to control diet 1-week post-surgery and then one group was switched to GZ667161 or kept on control diet 2-weeks post-surgery. Mice received weekly blood pressure and ECHO measurement between 4 and 7 weeks. At the end of 7 weeks post-surgery mice were euthanized for blood and tissue collection (Fig. 6A). At the reduced dose of GZ667161, we observed no significant changes or differences in body weight after recovery from CKD surgery between the 4 groups (Fig. 6B). As expected, relative to mice that received sham operation, CKD mice fed with control diet developed cardiac hypertrophy as reflected by increases in HW/TL and left ventricular myocyte area (Fig. 6C,D). CKD-induced cardiac dysfunction was blunted by feeding on GZ667161. Functional studies by ECHO revealed that CKD caused impairment in cardiac function in mice with control diet as evident by a decline in ejection fraction (Fig. 6E). CKD-induced cardiac functional decline was ameliorated by feeding CKD mice GZ667161 diet. At the reduced dose, GZ667161 also effectively inhibited GCS and reduced downstream GSL levels in in the plasma and heart tissue of mice ( Fig. 7A-H).
The effects on cardiac function and heart mass were further corroborated by studies on gene expression. As shown, CKD induced upregulation of BNP in mouse hearts fed on control diet (Fig. 8A). The increases in gene expression were blunted by feeding on GZ667161. CKD also increased TRPC6 expression cardiac fibrosis as evident by increased Col-I and -III expression and trichrome staining, which were blunted by GZ667161 diet (Fig. 8B-E). Overall, our results show that GZ667161 protects the heart against CKD-induced cardiac dysfunction. CKD-induced hypertension and biochemical disturbances may contribute to cardiac hypertrophy. To know whether the cardioprotective effect of GZ667161 may be related to these factors, we compared these parameters in CKD mice fed on control and GZ667161. As shown, clearance function reflected by degrees of azotemia (increased BUN level) was not different between CKD mice on control vs GZ667161 diets (Fig. 8F). We also found that water intake and urine output for CKD mice were increased compared to the sham controls for the respective groups but were not significantly different between the CKD groups (not shown). GZ667161 treatment did ameliorate CKD-induced increased urinary albumin excretion (Fig. 8G), but had no effect on CKD-associated increases in systolic blood pressure (Fig. 8H). CKD by 5/6 nephrectomy did not cause TRPC6 upregulation in the whole kidney tissue ( Supplementary Fig. 1A). TRPC6 upregulation is due to stress causes abnormal intracellular Ca 2+ increases [13][14][15]18 . As TRPC6 has NFAT-response element in the promoter, the initial abnormal intracellular Ca 2+ increases trigger a feedforward TRPC6 amplification and upregulation. We have found that lipid raft-associated PI3K is important for TRPC6 insertion to lipid rafts. GZ667161 ameliorates TRPC6 upregulation presumably by inhibiting lipid raft formation. As shown, in the absence of stress-induced upregulation, GZ667161 treatment had no effect on basal TRPC6 level in the kidney (Supplementary Fig. 1A). GZ667161 treatment prevented downregulation of klotho in CKD and ameliorated renal fibrosis in 5/6-nephrectomized remnant kidney tissues as shown by Col-I, Col-III, and trichrome staining ( Supplementary Fig. 1B-F).

Discussion
Glycosphingolipids (GSL) including cerebrosides, globoside, and gangliosides are important lipid constituents of the cell membrane. Genetic mutation of enzymes involved in GSL metabolism cause lysosomal storage diseases 35,36 . These diseases, such as Gaucher, Fabry's, Gangliosidosis, are results of cell/organ-toxicity from lipid overload. Enzyme replacement therapy is the mainstay therapy for these diseases. However, administered enzymes may not reach the organs to which they should target. Alternatively, the employment of substrate reduction therapy aims to reduce lipid overload in target organs by the use of small molecule drugs. Glucosylceramide synthase is the enzyme for the entry pathway for GSL synthesis. Many inhibitors of GCS (GCSi) including NB-DNJ (Miglustat), NB-DGJ (Lucerastat), Eliglustat and Venglustat, have been used or are under clinical trial for treating these diseases, supporting their safety profile and efficacy in clinical applications [37][38][39][40] .
Patients with CKD have an increased risk of developing cardiac hypertrophy despite adequate control of conventional risk factors such as hypertension, volume overload, and anemia. Previously we have shown in a mouse model of CKD that sKL deficiency is an important contributing factor 6 . TRPC6-mediated pathological calcium signaling plays an important role in feed-forward amplification of pathological cardiac remodeling. PI3K is essential for exocytotic insertion of TRPC6 channels 14,15,18 . Mechanistically, sKL targets to gangliosides in membrane lipid rafts to affect raft-associated PI3K and TRPC6 exocytosis to exert cardioprotection. Replacement by recombinant sKL is limited by the challenges of mass protein production. Thus, we tested whether pharmacological manipulation of membrane ganglioside content may be helpful for treatment of stress-induced cardiac dysfunction. Using two models of cardiomyopathy, isoproterenol overstimulation and a 5/6 nephrectomy CKD model, we showed that a newly developed GCSi, GZ667161, confers cardioprotection in both models. Compared to other GCSi such as, NB-DGJ (Lucerastat) which is currently in phase III clinical trial, GZ667161 inhibits sKL-mediated regulation of TRPC6 with ~ 50-fold higher potency. With respect to the effects on the kidney, GZ667161 treatment reduces fibrosis in the remnant tissue of 5/6-nephrectomized kidney. The protection against renal fibrosis may be related to amelioration of the downregulation of klotho in CKD. The detailed mechanism of how GZ667161 reduces klotho downregulation is unknown and remains to be investigated. GZ667161 treatment however does not affect CKD-induced urea clearance and CKD-associated hypertension. The possibility that GZ667161 affects other factors that impact cardioprotection, such as circulating FGF23 levels cannot be eliminated. Overall, the findings in the ISO-induced model supports the notion that GZ667161 can exert cardioprotective actions independently of renal protection.
Our study is the first to report the use of GCSi as an effective therapy without the presence of accumulation or abnormal levels of GSL in disease. In our study, GSL contents in the plasma and heart tissue are not increased in CKD or ISO-treated vs sham or non-treated mice. GCSi protects the heart by modulating GSL-dependent membrane signaling rather than correcting lipid overload. With regard to cardioprotection by GCSi, a previous study by Mishra   www.nature.com/scientificreports/ decreasing GSL overload and improving left ventricular mass and function in these mice 41 . Our study and the report by Mishra et al. strongly support the notion that decreasing cardiac gangliosides using GCSi confers protection against pathological cardiac hypertrophy. Our study demonstrates the efficacy of GCSi on stress-induced cardiomyopathy not originating from cardiac lipid overload and lipotoxicity. The proven safety profile of GCSi and higher potency raises the enthusiasm that GZ667161 or related GCSi may be a new therapy for causes of cardiac hypertrophy including cardiac hypertrophy in CKD patients. A recent study reported that mice with homozygous deletion of Ugcg encoding GCS in myocytes using α-MHC-Cre (Ugcg f/f ; α-MHC-Cre) developed dilated cardiomyopathy and premature death 23 . Of note, lipid rafts are ubiquitously present in eukaryotic cells. They are membrane platforms for growth factor receptors and associated signaling complexes, and for membrane trafficking 42 . The specific lipid raft type present in cardiomyocytes, caveolae, also play an important role in the ultrastructure of myocytes. α-MHC expression begins very early in embryonic development in mice, at around embryonic day 8.5 and increases at neonatal day 2 [43][44][45] . It is not surprising that deletion of GSL and lipid rafts early in development has detrimental effects in mice.
Oral administration of GCSi lowers GSL systemically and in many tissues as evident by the reduction in the plasma levels. While we show that GSL contents are reduced in the heart, we cannot exclude that cardioprotection occurs indirectly through other organs. Our results suggest that it is not through renal protection or improvement of CKD-induced hypertension. GZ667161 crosses the blood brain barrier and reduces GSL in CNS 31 . Thus, the possibility that an effect on CNS and neural control of heart function yet exists.
Lipid rafts are GSL-and cholesterol-enriched microdomains in which the lipid constituents are in dynamic equilibrium with those in the bulk membrane 42 . Formation of lipid rafts is governed by physicochemical properties of lipids and stabilized by local lipid-protein and protein-protein interactions 46 . The density of lipid rafts varies among cell types ranging from fractions of a percentage to a few percentages of total cell membrane 47 . www.nature.com/scientificreports/ As membrane platforms for signaling complexes and trafficking, lipid rafts are essential for cell and organism function. They are also targets for modulating cellular signaling cascades. The effect of sKL on raft-associated gangliosides does not result in an all-or-nothing function of lipid rafts in cells. Rather, sKL regulates the size and/or number of lipid rafts present. The abundance of gangliosides within the lipid raft increases when size of the lipid raft grows. This increases the local concentration of gangliosides and enhances the binding of sKL to gangliosides within rafts. sKL binding to gangliosides leads to dissociation and downsizing of lipid rafts. Thus, through regulating lipid raft formation sKL modulates raft-associated growth factor signaling. The physiological effect of sKL on cells and organs depends on lipid raft composition and association to signaling complexes of each cell type. The same principle applies to the pharmacological manipulation of lipid rafts by GCSi. As demonstrated in our pre-clinical studies, a therapeutic dose of GZ667161 is effective in cardioprotection and possesses a good safety profile. Aside from the cardiac stress-induced model, the baseline heart mass (i.e., -ISO or sham) in GZ667161-treated mice (while not statistically significant) tends to be lower than control fed mice (see Figs. 5B,E and 6C). Mouse organs continue to grow several months after birth. Through its effect on raft-associated growth factor signaling, GZ667161 may slow postnatal cardiac growth. We found that food intake is not different between feeding control vs GZ667161 diet on 0.011% GZ667161 but is different in 0.033% GZ667161 (not shown). Weight loss in mice that received a higher dose of GZ667161 may be due to this decrease in food intake but could also be related to similar effects on growth factor signaling. Whether these combined effects are applicable to adult human hearts is unknown. Nonetheless, it raises interesting questions to whether GCSi may be more broadly applicable in controlling non-stress-induced cardiac growth and hypertrophy.
In summary, this current study provides pre-clinical data that pharmacological inhibition of GCS by GZ667161 ameliorates ISO-induced and CKD induced cardiac dysfunction. This occurs in the setting of no overt lipotoxicity from GSL overload. The data presented provides robust in vivo evidence supporting a potential novel clinical therapeutic for cardiac dysfunction in CKD patients. The data are consistent with our working hypothesis that targeting gangliosides, as believed by sKL, is an effective therapeutic approach for cardioprotection.

Isoproterenol (ISO) induction model of cardiac hypertrophy.
Age-matched male 129/SvEv mice (Jackson Laboratory, Bar Harbor, ME, USA) of ~ 12 weeks old were used for ISO model experimentation. Mice were started on PicoLab® Rodent Diet 5053 (control diet) for 1 week before daily injections, and the drug groups were switched on 0.033% wt/wt GZ667161 diet (compound weight/food weight). Induction of cardiac hypertrophy was executed 3 days after diet switch by using ISO (3 mg/kg per day, diluted in normal saline) subcutaneous injection for 16 consecutive days. Blood pressure was taken before and after ISO injection by tail-cuff sphygmomanometer as previously described 18 . Echocardiogram (ECHO) to measure cardiac function was performed on day 17, then mice were weighed and euthanized for tissue collection using Isoflurane and cervical dislocation as approved by University of Iowa Institutional for Animal Care and Use Committee (IACUC). CKD model generated by 5/6 nephrectomy. CD-1 male mice (Charles River, Wilmington, MA, USA) ~ 10 weeks old were used for CKD model experiments. 5/6 nephrectomy surgery was performed as described before 6,49 . Mice were anesthetized with 5% isoflurane inhalation (AKORN Inc, Lake Forest IL, USA), and with continued 2% isoflurane inhalation flow throughout the surgery. The midabdominal region was cleaned with alcohol and sanitized with betadine solution. A ~ 2 cm incision was made to open up the abdomen. Using cotton tipped applicators, the left kidney was exposed and isolated. The upper and lower poles were cauterized, leaving approximately 1/3 of the left kidney. Then the right kidney was exposed. The right ureter and renal artery were tied off, and the right kidney was excised and removed. The surgical incision was sutured, and tissue glue was used to secure the closure. The mice were given 5 mg/kg of Carprofen (Zoetis Inc, Kalamazoo, MI, USA) for post-operative pain. Sham mice were generated by making and closing the incision without cauterization or removal of the kidneys. The mice were put on control diet 1-week post-operation. Week 2 post-operation one group of mice were left on control diet and another group was switched to 0.11% wt/wt GZ667161 diet. Blood pressure was measured, and urine was collected at Week 5 post-operation. ECHO was performed at Week 6 post-operation and mice were dissected for tissue collection.
Measurement of kidney function of CKD mice. Sham and CKD mice were placed unrestrained in metabolic cages for urine collection. 24-h and 48-h urine volume and water intake were measured by weight change of urine collection tubes or water bottles (g) converted to milliliters (mL). Urine was analyzed to examine albumin excretion, measured by albumin-creatinine ration (ACR), using QuantiChrom™ BCG Albumin and Creatinine Assay Kits (BioAssay Systems, Hayward, CA, USA). Blood was collected through retro-orbital bleed- General experimental procedure on mice. Systolic blood pressure and heart rate were measured noninvasively using tail cuff sphygmomanometer as previously described (Visitech BP-2000 Series IIBlood Pressure Analysis System) 18 . Echocardiogram imaging was performed using Acuson Sequoia model 256 clinical echocardiograph (Siemens, Malvern, PA,USA) fitted with an 8-MHz sector-scanning probe as described without the use of ketamine 50 . End-diastolic volume and stroke volume were measured in order to obtain ejection fraction percentage. Tissues (heart, tibia, and plasma) were collected at the end point. Body weight and heart weight were measured. Portions of heart tissue were collected for RNA isolation (saved in RNAlater), and lipid analysis (snap frozen and stored in -80 °C). The rest of the heart was stored in formalin for histological purposes. Tibia bones were digested with 1% SDS and 0.2 mg/kg proteinase K at 55ׄ°C overnight. Muscle and tissue were gently removed, and tibia was air-dried at room temperature. Tibia length was then measured using a digital caliper. All animal procedures were approved by IACUC and carried out in compliance with ARRIVE guidelines.
GSL analysis. Tissues utilized in this analysis were collected from ISO treated mice fed control or 0.033% GZ667161 Diet. Sphingolipid quantitative analysis was executed by liquid chromatography and high-resolution or tandem mass spectrometry (LC/HR-MS or LC/MS/MS) as previously described 51 . GlcCer and PC extractions, tissue homogenization protocol, analyses, and modifications for GM3 analysis were also conducted as previously detailed. PC was coextracted with sphingolipids for the purpose of normalization 51,52 . In order to extract GM3, approximately 10 µL of heart tissue homogenate (from approximately 100 mg heart tissue per sample) or 10 µL of serum plasma with a solvent comprised of equal volumes of acetonitrile and methanol. GM3 standard was purchased from Matreya, LLC. GM3 was separated with an isocratic elution using a mobile phase acetonitrile: methanol: water (61.5:33.23:5 vol/vol) in 10 mM of ammonium acetate, pH 6.8 for 2 min using a Waters Acquity UPLC and Waters Atlantis HILIC Silica column (3 mm, 2.1 × 100 mm) at room temperature. The flow rate was 0.4 mL/min. The eluent was analyzed by a Q Exactive mass spectrometer (ThermoFisher Scientific) that is equipped with a Heated ElectroSpray Ionization source. Data were gathered in full MS negative mode using the following parameters: scan range of 1100-1700 m/z, resolution 70,000, AGC target of 3e6, and max IT of 256 ns. Quantification of distinct isoforms with various acyl-chain lengths was exchanged by MS1 extraction of ion chromatograms 52 .
RNA isolation and RT-qPCR. RNA was extracted from heart tissue using Trizol (Invitrogen) Reagent.
RNA (10 mg) samples were then treated with TURBO DNase (Cat #AM1907, Invitrogen) prior to cDNA synthesis. cDNA for each sample was synthesized using 1 mg of RNA with iScript cDNA Kit (BioRad) performed in a BioRad Thermocycler. cDNA was diluted and RT-qPCR was performed in a 96 well-plate with SYBR Green PCR mix. The following primers were used: Histology. Hearts were stored and fixed in formalin at 4 °C and then paraffin embedded. Paraffin blocks were sectioned into 5 µm sections using Leica paraffin microtome in the Center of Microscopy at the University of Iowa. Sections were mounted onto Fischer brand frosted plus slides. Slides were then deparaffinized with rinses of xylene and stained with Masson's Trichrome staining for collagen (blue), muscle fibers (red), and nuclei (black/blue). Coverslips were then mounted on stained slides with Permount Mounting Medium. Slides were then scanned in slide scanner, PrimeHisto XE (Pacific Image Electronics, New Taipei City, Taiwan). For further magnification of cardiomyocytes, slides were viewed under compound light microscopy at 20X. Myocyte size and number were quantified using ImageJ (NIH, Bethesda, MD, USA).

Statistical analysis.
Statistical comparisons were made using student t-tests between control and experimental groups using GraphPad Prism. Each experiment was repeated at least once at different times with similar results. Data are presented as means ± SEM.
Ethics approval and consent to participate. The experimental protocols were approved and reviewed by University of Iowa Institutional for Animal Care and Use Committee (IACUC) and performed in conformity with ARRIVE guidelines.

Ethics declarations.
All animal studies were performed in accordance with the approved guidelines and protocols by University of Iowa Institutional for Animal Care and Use Committee (IACUC). Protocol #0,041,990.

Data availability
The datasets supporting the conclusions are included within this article or are available from the corresponding author upon request.